Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of cases are associated with shortening of the D4Z4 repeat sequences on chromosome 4q (FSHD1) while mutations in the SMCHD1 transcriptional repressor gene were linked to a smaller subset of FSHD patients (FSHD2). Mutations in SMCHD1 also greatly exacerbate the phenotype of FSHD1, thus acting as a modifier of the disorder's severity in FSHD1. Abnormal expression of the DUX4 gene present in the D4Z4 repeats is linked to the development of both FSHD1 and FSHD2. However, only a small percentage of patient muscle cells express DUX4 protein, which can also occasionally be observed in muscle cells from unaffected individuals. This suggests that DUX4 expression alone may not be sufficient for FSHD development. Thus, exactly how the DUX4 gene is upregulated in patient muscle cells and how it contributes to FSHD pathogenesis need to be further investigated. Since D4Z4 repeats are not present in the mouse genome, patient muscle cells are essential for assessing FSHD-specific cellular changes. However, high variability among samples with only a small subset of cells expressing DUX4 may exacerbate the averaging artifact of population analysis (Simpson's paradox). Though single-cell transcriptome analysis should address this conundrum, ?single-cell capture? is not optimal for large multinucleated myotubes. Thus, we propose to perform single-nucleus sequencing to determine the extent of cellular heterogeneity and test if a small population of cells carries the disease signature and drives FSHD pathogenesis. In Aim 1, we plan to perform single-nucleus RNA-sequencing to determine the extent of transcriptome heterogeneity in control, FSHD1 and FSHD2 myoblasts before and after differentiation. In Aim 2, we will focus on comparative analysis of DUX4- expressing and non-expressing cells in FSHD1 and FSHD2 to determine DUX4-dependent and ?independent changes in two types of FSHD. The goal of this study, therefore, is to define DUX4-mediated pathogenic changes in patient muscle cells and to understand the extent of heterogeneity of patient cell population and capture the possible ?disease-driving? cells. The project should have direct impact on our understanding of FSHD biology and potential development of novel diagnostic/therapeutic strategies and optimization of patient care.